Pig-a Evaluation in Mouse and MutaMouse Models Using Plate-Based Staining Method

UKEMS 2022 -- The in vivo Pig-a gene mutation assay is based on the endogenous X-linked Pig-a gene, which codes for an N-acetyl glucosamine transferase that is involved in the biosynthesis of Glycosyl Phosphatidyl Inositol (GPI) anchor for tethering a variety of mammalian cell surface protein markers. Pig-a gene mutation is quantified using flow cytometry to detect cells with either bound or unbound fluorescently tagged antibodies to GPI-tethered proteins as a single mutation in the Pig-a gene results in the loss of the extracellular GPI anchor structure. The Pig-a assay has been evaluated internationally and a draft OECD test guideline approved for publication (Mammalian Erythrocyte Pig-a Gene Mutation Assay, Dec 2021). The data generated to date indicate that it is sensitive, robust and has high reproducibility and transferability. Current guidance for the assessment of DNA reactive (mutagenic) impurities in pharmaceuticals (ICH M7(R1)) has already suggested the use of the Pig-a assay to investigate the in vivo relevance of in vitro mutagens (Ames positive). In a recent publication (Robinson TW, et al. 2021), it was suggested that combining the Pig-a end-point within a TGR study could assist in following up a positive Ames response. A 28-day dosing regimen was selected for this study to align with the OECD 488 standard TGR (transgenic rodent) study design. Following validation of the in vivo Pig-a assay in the rat at Labcorp, using the tube-based staining method, the primary aim of this study was to validate the 96 deep-well plate format of sample labelling and staining in the Mouse (CD-1 strain).